Zinc is an essential nutrient for all organisms but can be toxic if accumulated in excess amounts. Therefore, as extracellular zinc concentrations change, cells must maintain an adequate intracellular supply of zinc to meet metabolic demands while preventing its over accumulation. The long-range goal of this research is to understand the molecular mechanisms of cellular zinc homeostasis in eukaryotic cells. This process involves a complex interaction between zinc binding proteins and chelators, and the regulatory mechanisms that control these activities. Despite the importance of zinc to human health, we know little about zinc homeostasis. This proposal describes a combined genetic, molecular, biochemical, and biophysical approach to the study of zinc and zinc transporters. Their expression is induced in zinc deficiency by Zap1, a zinc-responsive transcriptional activator protein. Zap1 is proposed to be the intracellular zinc sensor for this regulation and its activity is controlled by Zn(II) binding directly to the protein to control its DNA binding activity. A second central hypothesis to the proposed research is that Zap1 target genes in addition to ZRT1 and ZRT2 play critical roles in zinc homeostasis. To test these hypotheses, the following specific aims are proposed: a) determine the in vivo effects of zinc on Zap1 DNA binding, b) identify potential accessory factors required for regulating Zap1 activity, c) use in vivo genetic analysis to identify residues in Zap1 that are required for zinc-responsive regulation, d) establish in vitro systems to study Zap1 regulation by Zn(II), e) map and characterize metal-binding sites in the Zap1 zinc-responsive domain in vitro, f) characterize the Zn(II)-induced molecular switch in the Zap1 zinc-responsive domain, g) define the role of the Zrc1, Cot1, and Zrt3 transporters in vacuolar zinc storage and detoxification, and h) characterize new transporters responsible for intracellular zinc transport and efflux. These studies should provide greater insight into the processes of zinc homeostasis in all eukaryotes.